Tryptophan as a functional replacement for ADP-ribose-arginine in recombinant proteins

ABSTRACT

A method is disclosed for producing a polypeptide with a modified activity or stability, by replacing an arginine residue capable of being ADP-ribosylated with a tryptophan or a phenylalanine. In one embodiment, compositions are provided that include polypeptides, such as alpha defensin, with arginine-to-tryptophan or arginine-to-phenylalanine substitutions, where the arginine residue is capable of being ADP-ribosylated. In another embodiment, methods are disclosed for modifying an immune response in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/517,565, filedDec. 7, 2004 now U.S. Pat. No. 7,541,139, which is the §371 U.S.National Stage of International Application No. PCT/US2003/020498, filedJun. 27, 2003, which was published in English under PCT Article 21(2),which in turn claims the benefit of U.S. Provisional Application No.60/393,033, filed Jun. 28, 2002, all of which are incorporated byreference herein in their entirety.

FIELD

The present disclosure relates generally to the modification of proteinsto alter protein activity and stability, specifically, to thesubstitution of phenylalanine or tryptophan for an arginine residuecapable of being adenosine-diphosphate (ADP)-ribosylated in apolypeptide sequence.

BACKGROUND

Mono-ADP-ribosylation of arginine residues in proteins is a reversiblemodification that involves the following steps: (i) the transfer of anADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) to anarginine residue of a target protein, or to a free arginine residue, byan arginine-specific ADP-ribosyltransferase (ART) and (ii) the cleavageof the bond between ADP-ribose and arginine by an ADP-ribosylargininehydrolase.

ARTs were first characterized in bacterial toxins, such as choleratoxin, diphtheria toxin, pertussis toxin, and pseudomonas exotoxin A.ADP-ribosyltransferase activity has since been identified in eukaryoticcells. The widespread expression of ARTs in eukaryotes, as well as inprokaryotes, suggests that the cycle ofADP-ribosylation/de-ADP-ribosylation of amino acid residues is widelyinvolved in regulating protein activity. Moreover, specific ADP-riboseacceptors, such as arginine, may serve as regulatory switches. Forexample, ADP-ribosylation of a specific arginine residue in thedinitrogenase enzyme of the nitrogen-fixing bacteria Rhodospirilliumrubrum has been shown to regulate the activity of this enzyme.

In eukaryotes, ART activity is linked to regulatory signals for criticalcellular processes such as DNA repair and the maintenance of calcium orphosphorylation levels. In humans, altered cellular ADP-ribosylationlevels have been linked to a number of diseases including lupus,diabetes and cancer, whereas bacterial toxins, such as cholera toxin anddiphtheria toxin, catalyze the ADP-ribosylation of important metabolicor regulatory proteins in their human hosts.

The ability to identify specific amino acids that can be modified inorder to regulate the activity of various proteins is critical in thedevelopment of medical treatments and therapies. Thus there is a need toidentify additional stable protein modifications that have an effect onprotein activity.

SUMMARY

Methods of producing a protein with an altered activity or stability aredisclosed herein. The method includes replacing an arginine residuecapable of being ADP-ribosylated with either a tryptophan (W) residue ora phenylalanine (F) residue, thereby producing a protein with anincreased activity or stability. In one embodiment, the protein has anantimicrobial activity and the stability or activity of the protein isincreased. In another embodiment, the protein is a defensin polypeptide.Substitution of an arginine capable of being ADP-ribosylated with eithera phenylalanine or a tryptophan results in an increased antimicrobialactivity of the defensin molecule or increased stability of the defensinmolecule. Specific, non-limiting examples of an antimicrobial activityare T cell chemotaxis, promotion of neutrophil recruitment, or cytokinerelease.

A method is provided for increasing the activity or stability of adefensin polypeptide comprising an arginine residue capable of beingADP-ribosylated. The method includes substituting the arginine residuewith a tryptophan or a phenylalanine, thereby increasing the activity orthe stability of the defensin polypeptide.

In another embodiment, a method is disclosed for determining if aprotein can be stabilized. The method includes determining if anarginine residue in the protein is capable of being ADP-ribosylated.Detection of ADP-ribosylation of the arginine residue indicates that thestability of the protein, such as a protein with antimicrobial activity,can be increased by substituting the arginine capable of beingADP-ribosylated with either a tryptophan or a phenylalanine.

A composition is disclosed herein that includes a polypeptide where atleast one arginine residue capable of being ADP-ribosylated issubstituted with a tryptophan or a phenylalanine residue. In oneembodiment, the protein has an antimicrobial activity. In anotherembodiment, the protein is a defensin polypeptide. In yet anotherembodiment, the amino acid substitution increases the activity orstability of the polypeptide.

A pharmaceutical composition is disclosed herein that includes atherapeutically effective amount of a defensin with at least onearginine residue capable of being ribosylated substituted by atryptophan or a phenylalanine residue.

In another embodiment, a method is provided for increasing an immuneresponse in a subject. The method includes administering to the subjecta therapeutically effective amount of a defensin polypeptide comprisingan amino acid substitution, wherein the amino acid substitution is areplacement of an arginine capable of being ADP-ribosylated with atryptophan or a phenylalanine.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic drawing of the deduced amino acid sequences of ratART2a (RT6.1) and rat ART2b (RT6.2). Identical amino acids are shaded.Arginine 204 and 81 are specific to ART2b. In ART2a, N58 and 58NKSE61are in a putative consensus glycosylation site not present in ART2b.Regions I, II, and III, believed to participate in formation of thecatalytic site in the bacterial toxin and mammalianADP-ribosyltransferases, are indicated by solid lines and the putativecatalytic amino acids by an asterisk. Dotted underlines indicate signalsequences, which are excised during the export into the endoplasmicreticulum (amino terminus) and attachment of theglycosylphosphotidylinositol (GPI) anchor (carboxy terminus).

FIG. 2 is a digital image of a set of blots demonstrating theauto-[³²P]ADP-ribosylation (FIG. 2A) and immunoreactivity (FIG. 2B) ofthe supernatants from NMU cells transfected with vector alone (lane 1),wild-type ART2b (lane 2), ART2b(R81K) (lane 3), ART2b(R204K) (lane 4),wild-type ART2a (lane 5), ART2a(M81R) (lane 6) and ART2a(Y204R) (lane7). Data shown are representative of eight independent experiments.

FIG. 3 is a digital image of a set of blots demonstrating theauto-ADP-ribosyltransferase activity (FIG. 3A) and immunoreactivity(FIG. 3B), as well as data demonstrating the NAD glycohydrolase (NADase)activity (nmol per hour) (FIG. 3C) of ART2b, ART2a, and various mutantforms of these proteins. The left column shows data for the followingwild-type and mutant ART2b proteins: ART2b (lane 1), ART2b(R204K) (lane2), ART2b(R81K) (lane 3), ART2b(R204Y) (lane 4), ART2b(R204E) (lane 5),ART2b(R204W) (lane 6), ART2b(R81K,R204K) (lane 7). The right columnshows data for the following wild-type and mutant ART2a proteins: ART2a(lane 1), ART2a(M81R) (lane 2), ART2a(Y204R) (lane 3), ART2a(M81R,Y204R)(lane 4), ART2a(N58A,Y204R) (lane 5), ART2a(59NMA61,Y204R) (lane 6).Data shown are representative of two experiments.

FIG. 4 is a digital image of a set of blots demonstrating theauto-ADP-ribosylation of ART2b (FIG. 4A), ART2a (FIG. 4B), and theirvarious mutant forms. The gels contain samples from cells expressingART2b wild-type (lane 1), ART2b(R81K) (lane 2), ART2b(R204W) (lane 3),ART2a(Y204R) (lane 4), ART2a(Y204R,M81R) (lane 5) andART2a(59NMA61,Y204R) (lane 6). Data represent one of two experiments.

FIG. 5 is a digital image of a set of blots demonstrating the SDS-PAGEseparation of auto-ADP-ribosylated ART2b and ART2b(R204K) proteins asanalyzed by a phosphorimager (FIG. 5A) and by immunoreactivity (FIG.5B). The blots contain samples from cells expressing wild type ART2b(lanes 1), ART2b(R204K) (lanes 2), or a mixtures of samples containingART2b and ART2b(R204K) (lanes 3). Samples were incubated with or without10 μM [³²P]NAD followed by either TCA precipitation or furtherincubation with 5 mM NAD. The radiolabeled wild type ART2b andART2b(R204K) proteins were combined and then incubated with or without 5mM NAD.

FIG. 6 is a digital image of a set of blots demonstrating the SDS-PAGEseparation of proteins tested for their sensitivity to acid,hydroxylamine and mercuric chloride. Samples from cells expressingwild-type ART2b, ART2b(R204W), ART2a(M81R,Y204R) and ART2a(N58A,Y204R)were auto-ADP-ribosylated with 10 μM[³²P]NAD followed by addition of 10%TCA (column II), or further incubation with 5 mM NAD at 30° C. for 1hour before precipitation with 10% TCA (column I). Neutralized sampleswere suspended in 0.1M Tris-HCl pH 7.5 (lane 1), 0.2M HCl (lane 2), 10mM HgCl₂ (lane 3), 2M NH₂OH (lane 4), or 0.2M NaCl (lane 5) for 2 hoursat 37° C. The samples were separated by SDS-PAGE in 12% gels,transferred to nitrocellulose and analyzed by phosphorImager (FIG. 6A)and by immunoblot with antipeptide antibody 1126 (FIG. 6B). Data arefrom one experiment, representative of two.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO:1 is the amino acid sequence of the human neutrophil peptide(HNP)-1, HNP-2, HNP-3 prepro-protein.

SEQ ID NO:2 is the amino acid sequence of HNP-1.

SEQ ID NO:3 is the amino acid sequence of HNP-2.

SEQ ID NO:4 is the amino acid sequence of HNP-3.

SEQ ID NO:5 is the amino acid sequence of the HNP-4 prepro-protein.

SEQ ID NO:6 is the amino acid sequence of HNP-4.

SEQ ID NO:7 is the amino acid sequence of the human defensin (HD)-5prepro-protein.

SEQ ID NO:8 is the amino acid sequence of HD-5.

SEQ ID NO:9 is the amino acid sequence of the HD-6 prepro-protein.

SEQ ID NO:10 is the amino acid sequence of HD-6.

SEQ ID NO:11 is the amino acid sequence of rat ART2a.

SEQ ID NO:12 is the amino acid sequence of rat ART2b.

SEQ ID NO:13 is the primer for introduction of Kozak sequence.

SEQ ID NO:14 is the primer for the ART2a N58A mutation.

SEQ ID NO:15 is the primer for the ART2a K59M, S60N, E61A mutation.

SEQ ID NO:16 is the primer for the ART2a M81R mutation.

SEQ ID NO:17 is the primer for the ART2a Y204R mutation.

SEQ ID NO:18 is the primer for the ART2b R81K mutation.

SEQ ID NO:19 is the primer for the ART2b R204K mutation.

SEQ ID NO:20 is the primer for the ART2b R204E mutation.

SEQ ID NO:21 is the primer for the ART2b R204Y mutation.

SEQ ID NO:22 is the primer for the ART2b R204W mutation.

DETAILED DESCRIPTION

I. Abbreviations

-   ADP adenosine-diphosphate-   ART ADP-ribosyltransferase-   ELISA enzyme-linked immunosorbent assay-   F phenylalanine-   GPI glycosylphosphatidylinositol-   HD human defensin-   HNP human neutrophil peptide-   IL interleukin-   M81R methionine-to-arginine substitution at position 81-   MIP macrophage inflammatory protein-   N58A asparagine-to-alanine substitution at position 58-   NAD nicotinamide adenine dinucleotide-   NADase nicotinamide adenine dinucleotide glycohydrolase-   PI-PLC phosphatidlyinositol specific phospholipase C-   R arginine-   R81K arginine-to-lysine substitution at position 81-   R204E arginine-to-glutamic acid substitution at position 204-   R204K arginine-to-lysine substitution at position 204-   R204Y arginine-to-tyrosine substitution at position 204-   R204W arginine-to-tryptophan substitution at position 204-   R:W arginine-to-tryptophan substitution-   R:F arginine-to-phenylalanine substitution-   W tryptophan-   Y204R tyrosine-to-arginine substitution at position 204-   59NMA61 asparagine, methionine and alanine at positions 59, 60 and    61, respectively    Standard one-letter codes for amino acids are utilized herein.    II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Activity: The biological function of a molecule, such as a polypeptideor a nucleic acid. In one embodiment, an activity is an enzymaticactivity. In another embodiment, a biological function is an immunologicactivity, such as recruitment of a cell, or cytokine secretion. Anactivity can be increased or decreased. An increased activity can be,for example, at least about a 20%, about a 50%, about an 80%, about a100% or about a 200% increase in activity. An activity can also bedecreased, such as at least about a 20%, about a 50%, about an 80% orabout a 100% decrease in activity. The activity can be increased ordecreased as compared to a control, such as the activity of a wildtypeprotein or a standard value. An activity profile is the set of differentactivities possessed by a molecule, such as an agent or a drug. Apolypeptide can have a single defined activity, or can have severaldefined activities.

The biological activity of a defensin molecule include modulating T cellchemotaxis and neutrophil recruitment. In one embodiment, an increasedantimicrobial activity of a defensin molecule includes increased T cellchemotaxis or increased neutrophil recruitment, as compared to a controldefensin molecule under similar conditions.

A specific, non-limiting example of the activity of an ART includes, butis not limited to, NADase activity. In one embodiment, an increasedactivity of an ART includes increased NADase activity, as compared to acontrol ART under similar conditions.

ADP-ribosylation: A reaction in which ADP-ribose is covalently attachedto a compound. Eukaryotic and prokaryotic mono-ARTs catalyze thetransfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) toan acceptor nucleophile, such as an amino acid (i.e. the guanidino groupof an arginine residue). Among the ARTs are bacterial toxins (e.g.cholera toxin, pertussis toxin, diphtheria toxin). Pertussis toxin anddiphtheria toxin use amino acids other than arginine as ADP-riboseacceptors.

As disclosed herein, a number of proteins used in host defense are basicand arginine-rich and thus could serve as acceptors for ADP-ribose.These include, but may not be limited to, alpha defensins (HNP-1, HNP-2,HNP-3, HNP-4, HD-5, HD-6); Beta defensins (hBD1, hBD-2, hBD-3, hBD-4);Major Basic Protein; Eosinophil Cationic Protein; and Human CathelicinLL-37 (hCAP18). In addition, ADP-ribosyltransferases are capable ofauto-ADP-ribosylation. These include, but may not be limited to, ART-1,ART2b, ART-3, ART-4, and ART-5.

ADP-ribosyltransferase (ART): An enzyme that catalyzes the transfer ofan ADP-ribose from NAD to an acceptor nucleophile. ARTs can bedifferentiated by their corresponding amino acid targets, which includearginine, cysteine, asparagine, and diphthamide (a post-translationallymodified histidine residue). In one embodiment, the ART catalyzes thetransfer of ADP-ribose to the guanidino group of an arginine residue ona protein.

Both prokaryotic and eukaryotic ARTs have been identified. Among theprokaryotic ARTs are bacterial toxins (e.g., cholera toxin, pertussistoxin, diphtheria toxin). Five mammalian ARTs (ART-1, ART-2, ART-3,ART-4, ART-5) are known to exist. Substrates of the five known mammalianARTs include proteins that are involved in critical cellular events(e.g., lymphocyte activation and neutrophil chemotaxis).

A family of mammalian ARTs that are localized on the cell surfacethrough glycosylphosphatidylinositol (GPI) anchors, are expressedpreferentially on epithelial and inflammatory cells (for examplelymphocytes and neutrophils). ART2a and ART2b are isoenzymes expressedon the surface of mature T cells and intraepithelial lymphocyte cells ofthe rat. These proteins express both auto-ART and NADase activities,although only ART2b is capable of auto-ADP-ribosylation at multiplesites. Of the two proteins, only ART2a is glycosylated. In addition,both are involved in the transmission of transmembrane signals thatmodulate T cell activation. Soluble forms of ART have also beenidentified and circulate in the high-density lipoprotein fraction ofserum.

Analysis of the crystallographic structure of bacterial toxin ARTsidentified three regions involved in formation of the catalytic site,NAD binding, and activation of the ribosyl-nicotinamide bond, which isrequired for ADP-ribose transfer. These regions appear to be present inthe mammalian transferases as well. Region I is defined by an arginine(R) or histidine (H), Region II, by a sequence rich in hydrophobic aminoacids, or by serine (S) X S, (where X represents threonine (T), serine(S) or alanine (A)), and Region III by glutamate (E). (Domenighini etal., Mol Microbial 21(4):667-74, 1996; Bredehorst et al., Adv Exp MedBiol 419:185-9, 1997; Moss et al., Mol Cell Biochem 193(1-2):109-13,1999; Takada et al., J Biol Chem (269(13):9420-3, 1994).

Agent: Any substance, including, but not limited to, a chemicalcompound, a drug, a small molecule, a peptide mimetic, a peptide or aprotein.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antimicrobial: A compound, such as an agent or a drug, for killingmicroorganisms or suppressing their multiplication or growth. An agenthas antimicrobial activity if it results in the death of a microorganismor suppresses the growth of a microorganism. In one embodiment, apolypeptide, such as a defensin (i.e. an alpha defensin), hasantimicrobial activity. An antimicrobial activity includes, but may notbe limited to, cell lysis (e.g. due to cytotoxicity). Antimicrobialactivity can result from T cell chemotaxis, and neutrophil recruitment.In one specific example, an antimicrobial activity is the lysis of abacterial cell. Antimicrobial activity can be modified by theadministration of a modified defensin polypeptide. In one embodiment, anR:W substituted, R:F substituted HNP-1 polypeptide, or otherwisemodified defensin, is administered to a subject.

Arginine (R): An amino acid (C₆H₁₄N₄O₂) found in plants and animals thatis essential for the human diet; also produced by the breakdown ofproteins. Also encompassed are functional analogues of arginine, andstructurally modified arginine molecules (e.g., ADP-ribosylated arginineresidues, agmatine) on a guanidine-containing compound, arginine beingone such example. An arginine residue that is capable of beingADP-ribosylated is an arginine that can be modified by the transfer ofan ADP-ribose from NAD to the guanidino group of an arginine.

Asthma: A disorder of the respiratory system characterized byinflammation, narrowing of the airways and increased reactivity of theairways to inhaled agents. Asthma is frequently, although notexclusively, associated with atopic or allergic symptoms.

B cell or B lymphocyte: One of the two major types of lymphocytes. Theantigen receptor on B lymphocytes, sometimes called the B cell receptor,is a cell-surface immunoglobulin. On activation by an antigen, B cellsdifferentiate into cells producing antibody molecules of the sameantigen-specificity as this receptor.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Chronic Bronchitis: An inflammation of the lining of the bronchi. Whenthe bronchi are inflamed and/or infected, less air is able to flow toand from the lungs and a heavy mucus or phlegm is coughed up, resultingin bronchitis. A brief attack of acute bronchitis with cough and mucusproduction can occur with severe colds. Chronic bronchitis is defined bythe presence of a mucus-producing cough most days of the month, threemonths of a year for two successive years without other underlyingdisease to explain the cough. It may precede or accompany pulmonaryemphysema. Cigarette smoking is the most common cause of chronicbronchitis. The bronchi of people with chronic bronchitis may also havebeen irritated initially by bacterial or viral infections. Air pollutionand industrial dusts and fumes are also potential etiologic agents. Oncethe bronchi have been irritated over a substantial period of time,excessive mucus is produced constantly, the lining of the bronchibecomes thickened, an irritating cough develops, airflow may behampered, and the lungs are damaged. The bronchi become susceptible toinfections.

Crohn's Disease: Crohn's disease is an Inflammatory Bowel Disease (thegeneral name for diseases that cause inflammation in the intestines).Crohn's Disease causes inflammation in the small intestine. Crohn'sDisease usually occurs in the lower part of the small intestine (theileum), but it can affect any part of the digestive tract, from themouth to the anus. The inflammation extends deep into the lining of theaffected organ, causing pain and diarrhea. Crohn's Disease may also becalled ileitis or enteritis.

Chronic Obstructive Pulmonary Disease (COPD): Includes emphysema andchronic bronchitis-diseases that are characterized by obstruction toairflow. Emphysema and chronic bronchitis frequently coexist. It doesnot include other obstructive diseases such as asthma.

Cytokines: Proteins made by cells that affect the behavior of othercells, such as lymphocytes and neutrophils. In one embodiment, acytokine is a chemokine, a molecule that affects cellular trafficking.Cytokines include, but are not limited to, MIP-β, interleukin (IL)-1,IL-8, IL-10, granulocyte-macrophage colony stimulating factor (GMCSF),granulocyte colony stimulating factor (GCSF), neurokinin, and tumornecrosis factor-alpha (TNF-α).

Defensins: The members of the defensin family are small, cationicpeptides that have six conserved cysteine residues that form threedisulfide bonds. Functional defensins arise by the sequentialpost-translational processing of a prepro-protein of 93-95 amino acids.The members of the defensin family are divided into different classes.The alpha-defensins are generally polypeptides containing 29-33residues. The beta-defensins are more basic than alpha defensins and aregenerally between 34-37 amino acids in length (Raj et al., Biochem J.347:633-41, 2000). The recently identified theta defensins are formed bythe head-to-tail linkage of two alpha defensin-related nonapeptides,generating a circular 18-residue polypeptide (Tang et al., Science286:498-502, 1999).

Defensins were first identified in neutrophils and have been detected inhuman, rabbit, guinea pig, and rat phagocytes. Alpha defensins include,but are not limited to, HNP-1, HNP-2, HNP-3, HNP-4, human defensin(HD)-5, and HD-6. Alpha defensins also include the recently identifiedHNP-4 homolog, defensin (Def)-X (see U.S. Pat. No. 6,329,340 hereinincorporated by reference). HNP-1, HNP-2, and HNP-3 are products of thesame gene (GenBank Accession No. P11479 herein incorporated byreference). HNP-4 is the product of a different gene (GenBank AccessionNo. NP_(—)001916 herein incorporated by reference). HD-5 (GenBankAccession No. NP_(—)066290) and HD-6, (GenBank Accession No.NP_(—)001917 herein incorporated by reference) are two human entericdefensins.

Defensins are antimicrobial peptides that are toxic for a variety ofinfectious agents, such as Gram-negative bacteria, Gram-positivebacteria, fungi, and certain enveloped viruses. Defensins act by formingpores in membranes of the infectious agent and generatingvoltage-dependent channels. Antimicrobial activities of defensinsinclude, but are not limited to, lysis of bacteria, fungi, or viruses;toxicity for bacteria, fungi or viruses; leukocyte (e.g., T cell)chemotaxis; and leukocyte (e.g., neutrophil) recruitment. Without beingbound by theory, defensins play an important role in the body's naturalimmunity against infections. Unmodified defensins are also cytotoxic forseveral normal and malignant cells.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer whichconstitutes the genetic material of most living organisms (some viruseshave genes composed of ribonucleic acid (RNA)). The repeating units inDNA polymers are four different nucleotides, each of which contains oneof the four bases, adenine, guanine, cytosine and thymine bound to adeoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequence of three nucleotides in the mRNA that istranscribed from the DNA.

Electrophoretic mobility: The relative distance that a molecule travelsin the presence of an electric current.

Emphysema: A condition in which there is over-inflation of structures inthe lungs known as alveoli or air sacs. This over-inflation results froma breakdown of the walls of the alveoli, which causes a decrease inrespiratory function and often, shortness of breath.

Encode: A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Functionally Equivalent: Sequence alterations, for example in anADP-ribosyltransferase2 (ART2) polypeptide that do not alter a functionof the ART2 polypeptide. In one embodiment, the function is themodulation of T cell activation. In another embodiment, the function isto modulate autoimmunity. Such sequence alterations can include, but arenot limited to, substitutions, deletions, base modifications, mutations,labeling, and insertions.

Immune cell: Any cell involved in a host defense mechanism. These caninclude, for example, T cells, B cells, natural killer cells,neutrophils, mast cells, macrophages, antigen-presenting cells,basophils, eosinophils, and neutrophils.

Immune response: A response of a cell of the immune system, such as aneutrophil, a B cell, or a T cell, to a stimulus. In one embodiment, theimmune response involves neutrophil recruitment, the phagocytosis of amicrobe by the neutrophil, followed by the release of the contents ofthe neutrophil's azurophilic granules. In another embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In yet another embodiment, an immune response is aninflammatory response. An immune response can be supplemented by theadministration of a modified defensin polypeptide. In one embodiment, anR:W substituted, R:F substituted HNP-1 polypeptide, or otherwisemodified defensin, is administered to a subject.

Immune system deficiency: A disease or disorder in which the subject'simmune system is not functioning normally, quantitatively orqualitatively, or in which it would be useful to boost a subject'simmune response. In another non-limiting example, the subject animmunodeficiency disease resulting from a human immunodeficiency virus(HIV) infection.

Infectious agent: An agent that can infect a subject and/or cause aninfection, including, but not limited to, viruses, bacteria, and fungi.

Examples of infectious virus include: Retroviridae (for example, humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III); Paramyxoviridae (for example,parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (for example, influenza viruses); andHerpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zostervirus, cytomegalovirus (CMV), herpes viruses).

Examples of infectious bacteria include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, Corynebacterium diphtheriae, Corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli.

Examples of infectious fungi include, but are not limited to,Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Other infectious organisms (such as protists) include: Plasmodiumfalciparum and Toxoplasma gondii.

Inflammation: When damage to tissue occurs, the body's response to thedamage is usually inflammation. The damage can be due to trauma, lack ofblood supply, hemorrhage, autoimmunity, transplanted exogenous tissue,or infection. This generalized response by the body includes the releaseof many components of the immune system (e.g. defensins, IL-1 and tumornecrosis factor), attraction of cells (such as neutrophils) to the siteof the damage, swelling of tissue due to the release of fluid, and otherprocesses.

During the inflammatory processes, a variety of soluble factors areinvolved in leukocyte recruitment through increased expression ofcellular adhesion molecules and chemoattraction. Many of these solublemediators regulate the activation of both the resident cells (such asfibroblasts, endothelial cells, tissue macrophages, and mast cells) andnewly recruited inflammatory cells (such as monocytes, lymphocytes,neutrophils, and eosinophils). In one embodiment, activated neutrophilsrelease azurophilic granules that contain defensins. High defensinlevels can be found in airway secretions of patients with inflammatorylung diseases.

Inflammatory Bowel Disease: Two separate diseases (Crohn's Disease andUlcerative Colitis) that cause inflammation of the bowel and can causearthritis or inflammation in joints. Crohn's Disease involvesinflammation of the colon or small intestines. Ulcerative Colitis ischaracterized by ulcers and inflammation of the lining of the colon. Theamount of the bowel disease usually influences the severity of arthritissymptoms.

Innate Immunity: Provides the first line of defense against many commonmicroorganisms and is essential for the control of common bacterialinfections. Includes antimicrobial peptides (e.g., defensins),epithelial barriers, phagocytic cells (neutrophils, macrophages),natural killer (NK) cells, the complement system, and cytokines thatregulate and coordinate many of the activities of these cells. Defensinpolypeptides are present at the surface of epithelial cells, such asthose lining the gut and the lungs, and in microbicidal organelles ofthe phagocytic cells of the hematopoietic system (e.g., neutrophils andmacrophages) and therefore are an important component to the innateimmune system. Innate immunity can be supplemented by the administrationof a modified defensin polypeptide. Thus, an R:W substituted, R:Fsubstituted HNP-1 polypeptide, or otherwise modified defensin, isadministered to a subject to increase innate immunity.

Isolated: A biological component (such as a nucleic acid, peptide orprotein) that has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins that have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cells, subdivided between 2 main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases or they may be recruitedto the site of infection.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B-cellsand T-cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Modified Arginine Residue: Any chemical modification of an arginine. Inone embodiment, the modification takes place on the guanidino group ofthe arginine residue. Modification of the guanidino group includes, butis not limited to, the modification of an arginine residue byADP-ribosylation, acylation, alkylation, or polymer conjugation. Anarginine residue that is ADP ribosylated can be further modified forexample, by the pyrophosphatase/phosphatase cleavage of a pyrophosphateto yield a ribosyl-arginine residue. In one embodiment, a decarboxylatedarginine residue is a modified arginine residue known as agmatine(C₅H₁₄N₄).

Neutrophil: Neutrophils are leukocytes of the PolymorphonuclearLeukocyte subgroup that are also known as granulocytes. Neutrophilscontain a lobed nucleus and abundant cytoplasmic granules that stainwith neutral dyes. Neutrophils form a primary defense against bacterialinfection. Like all the cells of the immune system, neutrophils areproduced in the bone marrow and circulate in the bloodstream. However,neutrophils move out of blood vessels into infected tissue in order toengulf and kill microorganisms (e.g., bacteria, fungus, virus).Neutrophils perform their function partially through the phagocytosis ofother cells and foreign substances. Neutrophils are recruited to a siteof infection by following a concentration gradient of chemoattractantsor cytokines.

Nicotinamide adenine dinucleotide glycohydrolase (NADase): An enzymethat catalyzes the hydrolysis of NAD⁺ to nicotinimide and ADP-ribose. Itis present ubiquitously in organisms from bacteria to mammals. NADasesfound in most eukaryotes are membrane bound and their release byphosphatidyl-inositol-specific phopholipase C suggests that they areanchored to the membrane via a GPI linkage.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide: A linear polynucleotide sequence of up to about 200nucleotide bases in length, for example a polynucleotide (such as DNA orRNA) which is at least 6 nucleotides, for example at least 15, 50, 100or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for a drug to interact with a cell. “Contacting” includesincubating a drug in solid or in liquid form with a cell.

A “therapeutically effective amount” is a quantity of a specificsubstance sufficient to achieve a desired effect in a subject beingtreated. For instance, this can be the amount necessary to alter animmune response and/or to inhibit viral, fungal, or bacterialreplication or to measurably alter symptoms of the viral, fungal, orbacterial infection. When administered to a subject, a dosage willgenerally be used that will achieve target tissue concentrations (forexample, in lymphocytes) that has been shown to achieve a desired invitro effect.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15^(th) Edition, 1975, describes compositions and formulationssuitable for pharmaceutical delivery of modified alpha defensins.

In general, the nature of the carrier will depend on the particular modeof administration employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Phenylalanine (F): An amino acid (C₉H₁₂NO₂) found in proteins.

Polynucleotide: A linear nucleotide sequence, including sequences ofgreater than 100 nucleotide bases in length.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. Either the L-opticalisomer or the D-optical isomer can be used, the L-isomers beingpreferred in nature. The term polypeptide or protein as used hereinencompasses any amino acid sequence and includes, but may not be limitedto, modified sequences including, but not limited to, substitutedpolypeptides, ADP-ribosylated polypeptides, ribosyl-polypeptides, andglycosylated polypeptides. The term polypeptide is specifically intendedto cover naturally occurring proteins, as well as those that arerecombinantly or synthetically produced.

Substantially purified polypeptide as used herein refers to apolypeptide that is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is for example, at least 80% free ofother proteins, lipids, carbohydrates or other materials with which itis naturally associated. In another embodiment, the polypeptide is atleast 90% free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. In yet anotherembodiment, the polypeptide is at least 95% free of other proteins,lipids, carbohydrates or other materials with which it is naturallyassociated.

Preventing or treating a disease: Preventing a disease refers toinhibiting completely or in part the development or progression of adisease, for example in a person who is known to have a predispositionto a disease. An example of a person with a known predisposition issomeone with a history of diabetes in the family, or who has beenexposed to factors that predispose the subject to a condition, such aslupus or rheumatoid arthritis. Treating a disease refers to atherapeutic intervention that ameliorates at least one sign or symptomof a disease or pathological condition, or interferes with apathophysiological process, after the disease or pathological conditionhas begun to develop.

Protein: A biological molecule encoded by a gene and comprised of aminoacids.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell.Preferably, a preparation is purified such that the protein or peptiderepresents at least 50% of the total peptide or protein content of thepreparation.

Pyrophosphatase: An enzyme that catalyzes the hydrolysis ofpyrophosphate into two phosphate groups.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or was made artificially. Artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques. Similarly, a recombinantprotein is one encoded by a recombinant nucleic acid molecule.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of a polypeptide, such as adefensin, and the corresponding cDNA sequence, will possess a relativelyhigh degree of sequence identity when aligned using standard methods.This homology will be more significant when the orthologous proteins orcDNAs are derived from species that are more closely related, comparedto species more distantly related (e.g., human and murine sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244 9, 1988);Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nuc. AcidsRes. 16:10881-90, 1988; Huang et al., Computer Appls. in the Biosciences8:155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994.Altschul et al., J. Mol. Biol. 215:403-410, 1990, presents a detailedconsideration of sequence alignment methods and homology calculations.

Stability: The ability of a substance, such as a polypeptide, tomaintain its form, structure or activity. Stability can be increased ordecreased. An increased stability is an increase in the ability of asubstance, such as a polypeptide, to maintain its form, structure oractivity, as compared to a control substance under similar conditions.In one embodiment, the stability of a polypeptide is increased by anamino acid substitution, such as an R:F or an R:W substitution, such asat least about a 20%, 50%, 80%, 100% or 200% increase, as compared to anunsubstituted polypeptide or to a wildtype polypeptide. Stability can bemeasured by any means known to one of skill in the part, and includes,but is not limited to, measurements of half-life.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

Substitution: The replacement of one amino acid residue with anotheramino acid residue using any technique known to one of ordinary skill inthe art, including site-directed mutagenesis of nucleic acid sequencesencoding the amino acid substituted polypeptide or chemical synthesis ofthe amino acid substituted polypeptide.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are non-limiting examples of amino acids thatare considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A non-conservative amino acid substitution can result from changes in:(a) the structure of the amino acid backbone in the area of thesubstitution; (b) the charge or hydrophobicity of the amino acid; or (c)the bulk of an amino acid side chain. Substitutions generally expectedto produce the greatest changes in protein properties are those inwhich: (a) a hydrophilic residue is substituted for (or by) ahydrophobic residue; (b) a proline is substituted for (or by) any otherresidue; (c) a residue having a bulky side chain, e.g., phenylalanine,is substituted for (or by) one not having a side chain, e.g., glycine;or (d) a residue having an electropositive side chain, e.g., lysyl,arginyl, or histadyl, is substituted for (or by) an electronegativeresidue, e.g., glutamyl or aspartyl.

Any cDNA sequence variant will preferably introduce no more than twenty,and preferably fewer than ten amino acid substitutions into the encodedpolypeptide. Variant amino acid sequences may, for example, be 80, 90 oreven 95% or 98% identical to the native amino acid sequence. Programsand algorithms for determining percentage identity can be found at theNCBI website.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is asuppressor T cell.

T cell chemotaxis: The directed locomotion of a T cell along aconcentration gradient of chemotactically active factors, such ascytokines. Cells showing positive chemotaxis move towards areas withhigher concentrations of these agents, those showing negative chemotaxismove away from these areas.

An increase in T cell chemotaxis includes, but may not be limited to, anincrease in the distance or rate of T cell migration, an increase in thenumber of T cells migrating, an increase in the types of T cellsmigrating in a sample in response to a chemotactic stimulus, as comparedto a control sample which does not receive the chemotactic stimulus.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease, such as pain or swelling.

Treatment: Refers to both prophylactic inhibition of initial infectionor disease, and therapeutic interventions to alter the natural course ofan untreated infection or disease process, such as a tumor growth or aninfection with a bacteria.

Tryptophan (W): An amino acid (C₁₁H₁₂N₂O₂) that is essential for growthand normal metabolism. Tryptophan is a precursor of niacin.

Ulcerative colitis: An Inflammatory Bowel Disease characterized byulcers and inflammation of the lining of the colon.

Wildtype: The form of a polypeptide or nucleic acid normally found innature. Also referred to as the native form. In one example, a wildtypepolypeptide is a polypeptide where an arginine residue that is capableof being ADP-ribosylated at a position within the amino acid sequence ofthe polypeptide has not been substituted.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Compositions and Administration of Pharmaceutical Compositions

A composition is provided herein that includes a polypeptide with anarginine-to-tryptophan (R:W) or an arginine-to-phenylalanine (R:F)substitution at a position within the amino acid sequence of thepolypeptide, wherein the arginine is capable of being ADP-ribosylated inthe unsubstituted form of the polypeptide. Substitution of an arginineresidue with a tryptophan (W) or a phenylalanine (F) residue yields apolypeptide with a modified activity and/or stability.

In one embodiment, a polypeptide includes a substitution of at leastone, at least two, at least three, or at least four arginine residuescapable of being ADP-ribosylated with a tryptophan or a phenylalanineresidue. The arginine residue that is capable of being ADP-ribosylatedcan be substituted with either a tryptophan or a phenylalanine at thisposition. In one specific, non-limiting example one arginine capable ofbeing ADP-ribosylated can be substituted with a tryptophan. In anotherspecific, non-limiting example, one arginine capable of beingADP-ribosylated can be substituted with a phenylalanine. In otherspecific, non-limiting examples two arginines capable of beingADP-ribosylated can be substituted with two tryptophans or twophenylalanines, or one tryptophan and one phenylalanine. Specific,non-limiting examples of a polypeptide with at least one arginineresidue capable of being ADP-ribosylated include a defensin or anADP-ribosyltransferase.

Polypeptides with R:W or R:F substitutions disclosed herein includepolypeptides with antimicrobial activity. Specific, non-limitingexamples of antimicrobial activity include the secretion of cytokines,chemotaxis of T cells, and neutrophil recruitment. In one embodiment,the polypeptide with antimicrobial activity is a defensin, such as analpha defensin.

In one specific embodiment, the alpha defensin is a vertebratepolypeptide, such as a mammalian polypeptide. In one example, the alphadefensin polypeptide is from a human. In other examples, the alphadefensin polypeptide is from a monkey, a rabbit, a rat, a cat, a dog, apig, a sheep, or a mouse. The alpha defensin can be a human neutrophilpeptide (HNP)-1. The alpha defensin polypeptide can also be HNP-2,HNP-3, HNP-4, HD-5, HD-6, or Def-X.

The alpha defensins include HNP-1, HNP-2, HNP-3, and HNP-4. HNP-1,HNP-2, and HNP-3 are products of the same 94 amino acid prepro-protein.The preproprotein has the sequence:

-   -   MRTLAILAAILLVALQAQAEPLQARADEVAAAPEQIAADIPEVVVS LAWDESLAPKH        PGSRKNMDCYCRIPACIAGERRYGTCIYQGRLWAFCC; (SEQ ID NO:1, see also        GenBank Accession No. P11479, herein incorporated by reference)        or a conservative variant thereof.

HNP-1 is one member of the family of alpha defensins produced bycleavage of the preproprotein. In one embodiment, HNP-1 has a sequenceas set forth as:

ACYCRIPACIAGERRYGTCIYQGRLWAFCC; (SEQ ID NO: 2)or a conservative variant thereof.

At least one of the arginine residues at positions 5, 14, 15, or 24(counting from the amino terminal end of the HNP-1 polypeptide sequence)of the HNP-1 polypeptide sequence as set forth as SEQ ID NO:2 is capableof being ADP-ribosylated. Thus, at least one of the arginine residues atposition 5, 14, 15, or 24 can be substituted with a tryptophan or aphenylalanine residue to produce an antimicrobial polypeptide withincreased stability and/or antimicrobial activity. Thus, in oneembodiment a tryptophan is included in the HNP-1 polypeptide in at leastone of positions 5, 14, 15, or 24. In another embodiment a phenylalanineis substituted for an arginine residue in at least one of positions 5,14, 15, or 24. In a further embodiment the HNP-1 polypeptide includesthe substitution of at least one tryptophan and at least onephenylalanine with an arginine that is capable of being ADP-rybosylated.

Specific, non-limiting examples of HNP-1 polypeptides with at least oneR:F or R:W substitution are shown in the table below.

TABLE 1 SEQ ID NO: 2 Position No.: 5 14 15 24 Native (SEQ ID NO: 2) R RR R Substitution W R R R Substitution R W R R Substitution R R W RSubstitution R R R W Substitution F R R R Substitution R F R RSubstitution R R F R Substitution R R R F Substitution W F R RSubstitution F W R R Substitution R R F W Substitution R R W FSubstitution R W F R Substitution R F W R Substitution F F F FSubstitution W W W W Substitution W W F F Substitution F F W WSubstitution F F F W Substitution F W F F Substitution F F W FSubstitution W F F F Substitution W W W F Substitution W W F WSubstitution W F W W Substitution F W W W Substitution W R R FSubstitution R W F R Substitution F R R W Substitution W R R F

HNP-2 is another member of the family of alpha defensins produced bycleavage of the preproprotein. In one embodiment, HNP-2 has a sequenceas set forth as:

CYCRIPACIAGERRYGTCIYQGRLWAFCC; (SEQ ID NO: 3)or a conservative variant thereof.

At least one of the arginine residues at positions 4, 13, 14, or 23(counting from the amino terminal end of the HNP-2 polypeptide sequence)of the HNP-2 polypeptide sequence as in SEQ ID NO:3 is capable of beingADP-ribosylated. Thus, at least one of the arginine residues atpositions 4, 13, 14, or 23 capable of being ADP-ribosylated can besubstituted with either a tryptophan or a phenylalanine residue toproduce an antimicrobial polypeptide with increased stability and/orantimicrobial activity. One of skill in the art can readily identifypolypeptides encompassed by the description set forth herein to generateexemplary substitutions similar to those shown in Table 2, below.

TABLE 2 SEQ ID NO: 3 Position No.: 4 13 14 23 Native (SEQ ID NO: 3) R RR R Substitution W R R R Substitution R W R R Substitution R R W RSubstitution R R R W Substitution F R R R Substitution R F R RSubstitution R R F R Substitution R R R F Substitution W F R RSubstitution F W R R Substitution R R F W Substitution R R W FSubstitution R W F R Substitution R F W R Substitution F F F FSubstitution W W W W Substitution W W F F Substitution F F W WSubstitution F F F W Substitution F W F F Substitution F F W FSubstitution W F F F Substitution W W W F Substitution W W F WSubstitution W F W W Substitution F W W W Substitution W R R FSubstitution R W F R Substitution F R R W Substitution W R R F

HNP-3 is a third member of the family of alpha defensins produced bycleavage of the preproprotein. In one embodiment, HNP-3 has a sequenceas set forth as:

DCYCRIPACIAGERRYGTCIYQGRLWAFCC; (SEQ ID NO: 4)or a conservative variant thereof.

At least one of the arginine residues at positions 5, 14, 15, or 24(counting from the amino terminal end of the HNP-3 polypeptide sequence)of the HNP-3 polypeptide sequence as in SEQ ID NO:4 is capable of beingADP-ribosylated. Thus, at least one of the arginine residues atpositions 5, 14, 15, or 24 is capable of being ADP-ribosylated and canbe substituted with either a tryptophan or a phenylalanine residue toproduce an antimicrobial polypeptide with increased stability and/orincreased antimicrobial activity. One of skill in the art can readilyidentify polypeptides encompassed by the description set forth herein togenerate exemplary substitutions, such as those shown in Table 3, below.

TABLE 3 SEQ ID NO: 4 Position No.: 5 14 15 24 Native (SEQ ID NO: 4) R RR R Substitution W R R R Substitution R W R R Substitution R R W RSubstitution R R R W Substitution F R R R Substitution R F R RSubstitution R R F R Substitution R R R F Substitution W F R RSubstitution F W R R Substitution R R F W Substitution R R W FSubstitution R W F R Substitution R F W R Substitution F F F FSubstitution W W W W Substitution W W F F Substitution F F W WSubstitution F F F W Substitution F W F F Substitution F F W FSubstitution W F F F Substitution W W W F Substitution W W F WSubstitution W F W W Substitution F W W W Substitution W R R F

HNP-4 is an alpha defensin that is the product of a prepro-proteinhaving a sequence as set forth as:

-   -   MRIIALLAAILLVALQVRAGPLQARGDEAGQEQRGPEDQDISISFAWDKS SALQVSG        STRGMVCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRVD (SEQ ID NO:5, see also        GenBank Accession No. NP_(—)001916, herein incorporated by        reference)        or a conservative variant thereof.

In one embodiment, HNP-4 has a sequence as set forth as:

VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRVD; (SEQ ID NO: 6)or a conservative variant thereof.

At least one of the arginine residues at positions 5, 10, 11, 15, or 32(counting from the amino terminal end of the HNP-4 polypeptide sequence)of the HNP-4 polypeptide sequence is capable of being ADP-ribosylated.Thus, at least one of the arginine residues at positions 5, 10, 11, 15,or 32 is capable of being ADP-ribosylated and can be substituted witheither a tryptophan or a phenylalanine residue to produce anantimicrobial polypeptide with increased stability and/or antimicrobialactivity. One of skill in the art can readily identify polypeptidesencompassed by the description set forth herein to generate exemplarysubstitutions similar to those shown in Tables 1, 2, or 3.

HD-5 is produced by cleavage of the following prepro-protein having asequence as set forth as:

-   -   MRTIAILAAILLVALQAQAES LQERADEATTQKQSGEDNQDLAISFAGNGLSALRTS        GSQARATCYCRTGRCATRESLSGVCEISGRLYRLCCR; (SEQ ID NO:7, GenBank        Accession No. NP_(—)066290, herein incorporated by reference)        or conservative variants thereof.

In one embodiment, HD-5 has a sequence as set forth as:

TCYCRTG RCATRESLSG VCEISGRLYR LCCR; (SEQ ID NO: 8)or conservative variants thereof.

At least one of the arginine residues at positions 5, 8, 12, 24, 27, or31 (counting from the amino terminal end of the HNP-5 polypeptidesequence) of the HNP-5 polypeptide sequence as in SEQ ID NO:8 is capableof being ADP-ribosylated. Thus, at least one of the arginine residues atpositions 5, 8, 12, 24, 27, or 31 is capable of being ADP-ribosylatedand can be substituted with a tryptophan or a phenylalanine residue toproduce an antimicrobial polypeptide with increased stability orantimicrobial activity. One of skill in the art can readily identifypolypeptides encompassed by the description set forth herein to generateexemplary substitutions similar to those shown in Tables 1, 2, or 3.

HD-6 is produced by cleavage of the following prepro-protein having asequence as set forth as:

-   -   MRTLTILTAVLLVALQAKAEPLQAEDDPLQAKAYEADAQEQRGANDQDFAVSFAE        DASSSLRALGSTRAFTCHCRRSCYSTEYSYGTCTVMGINHRFCCL; (SEQ ID NO:9,        GenBank Accession No. NP_(—)001917, herein incorporated by        reference)

In one embodiment, HD-6 has a sequence as set forth as:

TCHCRRSCYS TEYSYGTCTV MGINHRFCCL; (SEQ ID NO: 10)or a conservative variant thereof.

At least one of the arginine residues at positions 5, 6, or 26 (countingfrom the amino terminal end of the HNP-6 polypeptide sequence) of theHNP-6 polypeptide sequence as in SEQ ID NO:10 is capable of beingADP-ribosylated. Thus, at least one of the arginine residues atpositions 5, 6, or 26 is capable of being ADP-ribosylated and can besubstituted with a tryptophan or a phenylalanine residue to produce anantimicrobial polypeptide with increased stability and/or antimicrobialactivity. One of skill in the art can readily identify polypeptidesencompassed by the description set forth herein to generate exemplarysubstitutions similar to those shown in Tables 1, 2, or 3.

Any ADP-ribose acceptor that contains an arginine residue capable ofbeing ADP-ribosylated can be substituted with a tryptophan or aphenylalanine residue to produce an antimicrobial polypeptide withincreased stability and/or antimicrobial activity. In one embodiment, apolypeptide with an R:W or an R:F substitution, where the arginineresidue is capable of being ADP-ribosylated, is a polypeptide withNADase activity. In another embodiment, a polypeptide with an R:W or anR:F substitution, where the arginine residue is capable of beingADP-ribosylated, is a polypeptide with ART activity, such as an ART. Twospecific, non-limiting examples of a polypeptide with ART activityinclude ART2a and ART2b. Other specific, non-limiting examples ofpolypeptides with ART activity include, but may not be limited to, ART1,ART3, ART4, or ART5. In one embodiment, ART is a vertebrate polypeptide.In another embodiment, ART is a mammalian polypeptide. In yet anotherembodiment, ART is a rat polypeptide, for example ART2b.

The polypeptides disclosed herein can be produced by any method known toone of skill in the art. In one embodiment, the polypeptide issynthetic. Synthetic polypeptides having fewer than about 100 aminoacids, or fewer than about 50 amino acids, and can be generated usingknown techniques. For example, solid phase techniques, such as theMerrifield solid-phase synthesis method (Merrifield, J. Am. Chem. Soc.85:2149-2146, 1963), can be used to generate synthetic polypeptides.Equipment for automated synthesis is commercially available (e.g. PerkinElmer, Applied BioSystems). These automated synthesizers can be used toproduce substitutions (such as an R:W or R:F substitution) of thepeptide sequence of interest.

Polypeptides including an R:W or an R:F substitution of an arginine wascapable of being ADP-ribosylated can be produced recombinantly using aDNA sequence that encodes the polypeptide, which can be inserted into anappropriate expression vector. Methods are known to construct expressionvectors encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. In addition, methodsare known to one of skill in the art that are of use to producesite-directed mutations in a nucleic acid sequence of interest, suchthat translation of the sequence includes R:W or R:F substitution.Materials can also be synthesized chemically without requiringrecombinant DNA. This is especially true, but not limited to, smallpeptides or other arginine-containing compounds as noted above.

Method of Producing a Polypeptide with Modified Activity and/orStability

A method is provided herein to produce a polypeptide with modifiedactivity and/or stability. The method includes substituting an arginineresidue that is capable of being ADP-ribosylated, with a tryptophan or aphenylalanine residue in the amino acid sequence of the polypeptide. Inone embodiment, the polypeptide is an antimicrobial polypeptide.Specific, non-limiting examples of an antimicrobial polypeptide are adefensin or an ADP-ribosyltransferase. The method to produce apolypeptide with modified stability or activity can include substitutingat least one, at least two, at least three, or at least four arginineresidues that are capable of being ADP-ribosylated with a tryptophan ora phenylalanine residue within the amino acid sequence of a polypeptide.Thus, in one example a polypeptide can be produced in which with onearginine capable of being ADP-ribosylated is substituted with atryptophan. In another example a polypeptide can be produced in whichone arginine capable of being ADP-ribosylated is substituted with aphenylalanine. In other examples two arginines capable of beingADP-ribosylated are substituted with two tryptophans or twophenylalanines. In a further example at least one arginine capable ofbeing ADP-ribosylated is substituted with a phenylalanine and at leastone arginine capable of being ADP-ribosylated is substituted with atryptophan.

In one embodiment, the method produces a polypeptide with increasedactivity, compared to a control polypeptide, by making an R:Wsubstitution or an R:F substitution of an arginine capable of beingADP-ribosylated. In another embodiment, the method produces apolypeptide with a decreased activity of the polypeptide, compared to acontrol polypeptide. In one example, the increased activity is anenzymatic activity such as, but not limited to, NADase activity. Inother examples, the increased activity is ART activity, recruitment ofan immune cell, cytokine secretion, or an antimicrobial or cytotoxicactivity. The activity (for example the antimicrobial activity or thelysis of a pathogen in response to administration of the protein) can beincreased by at least about 20%, at least about 50%, at least about 80%,or at least about 100%. In another embodiment, the activity can bedecreased by at least about 20%, at least about 50%, at least about 80%,or at least about 100%.

A method is also provided for producing a polypeptide with increasedstability. The method includes producing a polypeptide with an R:W or anR:F substitution of an arginine capable of being ADP-ribosylated. Aspecific, non-limiting example of a control polypeptide is the wildtypepolypeptide including an arginine at the position of interest where thearginine is unsubstituted or ADP-ribosylated. Another example of acontrol is a standard value. In several embodiments, the increase instability can be at least about a 20%, at least about a 50%, at leastabout an 80%, or at least about a 100% increase in stability.

A substitution of an amino acid residue within a polypeptide, such asthe substitution of an arginine residue capable of beingADP-ribosylated, with a tryptophan or a phenylalanine residue, can beaccomplished by any means known to one of skill in the art. As describedabove, either genetic engineering or chemical synthesis techniques canbe used. In one specific, non-limiting example, standard DNA mutagenesistechniques include oliogonucleotide and PCR-mediated site-directedmutagenesis. Details of these techniques are provided in Sambrook et al.(In Molecular Cloning: A Laboratory Manual, CSHL, New York, 2001), Ch.13. In addition, as described above, amino acid substitutions can beintroduced by the chemical synthesis of molecules with the desired aminoacids at the specified residue position.

Method of Screening

Disclosed herein are methods for screening a polypeptide to determine ifthe polypeptide can be stabilized or if the activity of the polypeptidecan be altered. In one embodiment, the ability of an arginine residue tobe ADP-ribosylated is an indication that the polypeptide can bestabilized or that the activity of the polypeptide can be increased. Inone example, the polypeptide can be an antimicrobial polypeptide such asa defensin. The polypeptide can alternatively be an enzyme, such as anADP-ribosyltransferase.

One skilled in the art can readily determine if an arginine isADP-ribosylated. In one specific, non-limiting example, the polypeptideis incubated with an ART capable of ADP-ribosylating an arginineresidue. The ability of ART to ADP-ribosylate the polypeptide is thenassessed. The polypeptide can have at least one, such as at least two,at least three, or at least four, arginine residues that are capable ofbeing ADP-ribosylated. Any assay can be used to assess ADP-ribosylationsuch as, but not limited to, measurements of the electrophoreticmobility of the polypeptide, compared to that of a control polypeptide.A decrease in the electrophoretic mobility of the polypeptide, comparedto the control polypeptide, is an indication that the polypeptide isADP-ribosylated. A specific, non-limiting example of a controlpolypeptide is a polypeptide known not to be ADP-ribosylated, such as apolypeptide that has been incubated with a buffer (in place of the ART).Another specific, non-limiting example of a control polypeptide is apolypeptide with an R:F and/or an R:W substitution, wherein the arginineis not capable of being ADP-ribosylated.

In order to confirm that the polypeptide identified in the screeningmethod has altered stability or activity, the arginine residue issubstituted with a tryptophan or a phenylalanine. At least one, at leasttwo, at least three, or at least four arginine residues can besubstituted with a tryptophan or a phenylalanine residue and it can bedetermined if this polypeptide has a change in stability or activity. Inone embodiment, the stability of a polypeptide is increased by an aminoacid substitution, such as an R:F or an R:W substitution, such as atleast about a 20%, 50%, 80%, 100% or 200% increase, as compared to anunsubstituted polypeptide or to a control polypeptide. Stability can bemeasured by any means known to one of skill in the part, and includes,but is not limited to, measurements of half-life (t½) of thepolypeptide. The activity of the polypeptide with the R:W or the R:Fsubstitution can also be measured and compared to the stability of acontrol polypeptide. A specific, non-limiting example of a control is apolypeptide that has an arginine at one or more positions of interest,or a standard value.

A polypeptide with at least one, at least two, at least three, or atleast four R:W or R:F substitutions, can be tested to determine if theyhave an altered activity. In one embodiment, the activity of apolypeptide is increased by an amino acid substitution, such as an R:For an R:W substitution, such as at least about a 20%, 50%, 80%, 100% or200% increase, as compared to ac control polypeptide, such as anunsubstituted polypeptide. In another embodiment, the activity of apolypeptide is decreased by an amino acid substitution, such as an R:For an R:W substitution, such as at least about a 20%, 50%, 80%, or 100%decrease, as compared to a control polypeptide, such as an unsubstitutedpolypeptide, or a standard value.

Activity can be measured by any means known to one of skill in the art,and includes, but is not limited to, an enzymatic activity or animmunologic activity. Assays to measure enzymatic activity includekinase assays (such as serine/threonine or tyrosine kinase assays),autophosphorylation assays, phosphatase assays, NADase assay,ADP-ribosyltransferase assays, phosphodiesterase assays, glutamic aciddecarboxylase assays, oxygenoase assays. Assays to measure immunologicactivity include chemotaxis assays, cytokine production and secretionassays, biological assays for T-cell activity including assays forcytotoxic activity, Th1 activity and Th2 activity, assays for neutrophilrecruitment, and assays to measure B-cell activation. Other enzymaticand immunologic assays are known to those of skill in the art.

Pharmaceutical Compositions and Methods of Using SubstitutedPolypeptides

As disclosed herein, the amino acid substitution of an arginine residuethat is capable of being ADP-ribosylated can alter the activity of thepolypeptide or can alter the stability of the polypeptide. For example,the arginine-to-tryptophan (R:W) substitution or anarginine-to-phenylalanine (R:F) substitution in an antimicrobialpeptide, such as a defensin molecule, can increase the antimicrobialactivity of the peptide and/or modify an immune response in a subjectwhen a therapeutically effective amount is administered to a subject.Thus, a method is provided herein for modulating an immune responseagainst a microbe.

Defensin polypeptides are antimicrobial peptides that are involved ininnate immune defense and are cytotoxic for microbes such as bacteria,fungi, and certain types of viruses. In addition, they stimulate IL-8release from neighboring cells and induce an increase in T cellchemotaxis. As disclosed in the methods described above, the activity ofa defensin molecule can also be altered by substituting an arginineresidue with a tryptophan or a phenylalanine residue, where the arginineresidue is a residue that is capable of being ADP-ribosylated. Moreover,the R:W and/or R:F substituted defensin molecule can have an increasedantimicrobial activity compared to the defensin molecule with anADP-ribosylated arginine residue. The activity profile of the defensinmolecule can also be altered. The R:W and/or R:F substituted defensinmolecule can have an increased stability compared to a defensin moleculethat includes an ADP-ribosylated arginine residue.

In one embodiment, substituting an arginine residue with a tryptophan ora phenylalanine residue, where the arginine residue is capable of beingADP-ribosylated, increases the anti-microbial activity of the defensin,compared to an unsubstituted defensin molecule when administered to asubject. In several specific, non-limiting examples the alpha defensinincludes, but is not limited to, HNP-1, HNP-2, HNP-3, and HNP-4. Theantimicrobial activity can be antibacterial, antifungal, or antiviralactivity. In several specific, non-limiting examples, the increase inantimicrobial activity is at least about 20%, at least about 50%, atleast about 80%, at least about 100%, or at least about 200%. In anotherembodiment, the increased antimicrobial activity is an increase incytokine production. The increase in cytokine expression can be anincrease in cytokine secretion, expression, and/or release. In onespecific, non-limiting example, the cytokine is IL-8. The antimicrobialactivity can be an increase in recruitment of inflammatory cells, suchas neutrophils. Neutrophil recruitment can be measured by any methodknown to one of skill in the art. In one embodiment, promotion ofneutrophil recruitment is measured by the release of IL-8 from cells. Inone specific, non-limiting example, IL-8 release is measured by indirectenzyme-linked immunosorbent assay (ELISA).

In yet another embodiment, the altered antimicrobial activity is anincrease in inflammatory cell chemotaxis. In one specific, non-limitingexample the inflammatory cells are T cells.

Thus, a method of modifying an immune response against a microbe isprovided. The method includes administering a therapeutically effectiveamount of a defensin with an R:W substitution and/or an R:Fsubstitution, where the arginine residue is capable of beingADP-ribosylated, to a subject infected with or at risk of being infectedwith the microbe, thereby modulating the immune response against themicrobe. The immune response in the subject is increased, as compared toa subject treated with an unsubstituted defensin molecule, or anuntreated subject.

In one embodiment, modification of an immune response includesincreasing lymphocyte chemotaxis. Thus, the administration of atherapeutically effective amount of a defensin to a subject, such as analpha defensin with an R:W substitution or an R:F substitution where thearginine is capable of being ADP-ribosylated, modulates T cellchemotaxis in the subject. For example, T-cell chemotaxis is increasedin a subject following substitutions of a modified alpha defensin,compared to an unsubstituted alpha defensin molecule. T cell chemotaxiscan be measured by any means known to one of skill in the art, but isgenerally measured by measuring the length of migration of the T cells,the number of migrating T cells, or both. In one specific, non-limitingexample, T cell migration is measured in vitro, such as by measuring Tcell migration from one cell culture chamber to another cell culturechamber through a porous membrane.

In another embodiment, modification of the immune response includesaltering an inflammatory response. Thus, the administration of atherapeutically effective amount of a defensin, such as an alphadefensin, results in an increase in an inflammatory response. Aninflammatory response can be measured by any means known to one of skillin the art. In one embodiment, an inflammatory response is measured byassessing the number of activated T cells present in the sample. Inanother embodiment, an inflammatory response is measured by a change inneutrophil recruitment. In yet another embodiment, an inflammatoryresponse is measured by cytokine production and/or release, such as achange in IL-8 production and/or release. In several embodiments,increased cytokine production and/or release is a 100%, 200%, or 300%increase in cytokine production and/or release in the presence of adefensin with an R:W substitution or an R:F substitution, where thearginine residue is capable of being ADP-ribosylated, compared to acontrol.

The subject can be any mammal. In one embodiment, the subject is ahuman. In other embodiments, the subject may be a monkey, a rabbit, arat, a pig, a sheep, a dog, a cat, or a mouse. In one embodiment, thesubject is suffering from a disease, such as a pulmonary disease.Specific, non-limiting examples of pulmonary diseases are emphysema,adult respiratory distress syndrome, asthma, bronchopulmonary dysplasia,chronic bronchitis, sarcoidosis, pulmonary fibrosis, or cystic fibrosis.In another embodiment, the subject is infected with a pathogen, such asa bacteria, fungus, or virus. Specific, non-limiting examples ofbacterial infections affecting the lungs are pneumonia or tuberculosis.

In another embodiment, the subject has a tumor, such as a benign or amalignant tumor. Specific, non-limiting examples are lung, intestine,colon, breast, ovarian, uterine, prostate, testicular, or liver tumors.

In a further embodiment, the subject has an intestinal disease.Specific, non-limiting examples of intestinal diseases are inflammatorybowel diseases such as Crohn's Disease and ulcerative colitis.

In yet another embodiment, the subject is immunodeficient. In onespecific, non-limiting example, the subject is infected with animmunodeficiency virus, such as a human immunodeficiency virus (e.g.,HIV-1 or HIV-2). In a further embodiment, the subject has an autoimmunedisorder.

Pharmaceutical compositions can include a therapeutically effectiveamount of a polypeptide with an R:W or an R:F substitution, where thearginine residue is capable of being ADP-ribosylated, and can beformulated with an appropriate solid or liquid carrier, depending uponthe particular mode of administration chosen. Specific, non-limitingexamples of polypeptides with an R:W or an R:F substitution that have analtered activity or stability include alpha defensin and ART. Thepharmaceutically acceptable carriers and excipients useful in thisdisclosure are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable fluid vehicles such as water, physiologicalsaline, other balanced salt solutions, aqueous dextrose, glycerol or thelike. Excipients that can be included are, for instance, other proteins,such as human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered can also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

Other medicinal and pharmaceutical agents, for instance otherimmunostimulants, also can be included. Immunostimulants include, butare not limited to, Macrophage Inflammatory Protein (MIP)-β, IL-1, IL-8,IL-10, granulocyte-macrophage colony stimulating factor, granulocytecolony stimulating factor, neurokinin, and tumor necrosis factor-alpha,for example.

The dosage form of the pharmaceutical composition will be determined bythe chosen mode of administration to the subject. For instance, inaddition to injectable fluids, topical, inhalation, oral and suppositoryformulations can be employed. Topical preparations can include eyedrops, ointments, sprays and the like. Inhalation preparations can beliquid (e.g., solutions or suspensions) and include mists, sprays andthe like. Oral formulations can be liquid (e.g., syrups, solutions orsuspensions), or solid (e.g., powders, pills, tablets, or capsules).Suppository preparations can also be solid, gel, or in a suspensionform. For solid compositions, conventional non-toxic solid carriers caninclude pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

The pharmaceutical compositions that include a therapeutically effectiveamount of a polypeptide, such as an alpha defensin, with an R:W or anR:F substitution, where the arginine residue is capable of beingADP-ribosylated, can be formulated in unit dosage form, suitable forindividual administration of precise dosages. In one specific,non-limiting example, a unit dosage can contain from about 1 ng to about1 mg of such a polypeptide. The amount of active compound(s)administered will be dependent on the subject being treated, theseverity of the affliction, and the manner of administration, and, forexample, can be determined at the judgment of the prescribing clinician.Within these bounds, the formulation to be administered will contain aquantity of the active component(s) in amounts effective to achieve thedesired effect in the subject being treated.

The compounds of this disclosure can be administered to humans or otheranimals on whose tissues they are effective in various manners such astopically, orally, intravenously, intramuscularly, intraperitoneally,intranasally, intradermally, intrathecally, subcutaneously, viainhalation or via suppository. The particular mode of administration andthe dosage regimen will be selected by the attending clinician, takinginto account the particulars of the case (e.g. the subject, the disease,the disease state involved, and whether the treatment is prophylactic).Treatment can involve daily or multi-daily doses of compound(s) over aperiod of a few days to months, or even years during the course oftreatment. However, the effective amount of the defensin is dependent onthe subject being treated, the severity and type of the affliction, andthe manner of administration of the therapeutic(s).

A therapeutically effective amount of a polypeptide, such as an alphadefensin, with an R:W or an R:F substitution, of an arginine residuecapable of being ADP-ribosylated, can be the amount of a polypeptidenecessary to modulate the immune system of a subject. In severalexamples, a therapeutically effective amount is an amount of thesubstituted defensin sufficient to stimulate antimicrobial activity,such as an amount sufficient to stimulate T cell chemotaxis or promoteneutrophil recruitment.

Site-specific administration of the disclosed compounds can be used, forinstance by applying the amino acid substituted defensin polypeptide(for example an R:W or an R:F substituted alpha defensin, of an arginineresidue capable of being ADP-ribosylated) to a region of inflammation, aregion of infection, or a region suspected of being prone toinflammation or infection.

The present disclosure also includes combinations of a polypeptide, suchas an alpha defensin, with an R:W or an R:F substitution, of thearginine residue capable of being ADP-ribosylated, with one or moreother agents useful in the treatment of an immune-related disorder,condition, or disease. For example, the compounds of this disclosure canbe administered in combination with effective doses of immunostimulants,anti-cancer agents, anti-inflammatory agents, anti-infectives, and/orvaccines. The term “administration in combination” or“co-administration” refers to both concurrent and sequentialadministration of the active agents. A subject that is infected with aninfectious agent, or displays an immune suppression, will be a candidatefor treatment using the therapeutic methods of the disclosed herein, asdescribed below.

Additional Methods

A method is disclosed herein for inhibiting the cytotoxic activity of anative alpha defensin polypeptide in a subject. The method includesadministering to a subject a therapeutically effective amount of adefensin polypeptide with an arginine-to-tryptophan (R:W) or anarginine-to-phenylalanine (R:F) substitution, where the arginine residueis capable of being ADP-ribosylated, to decrease the cytotoxic activityof the polypeptide. In one embodiment, the defensin polypeptide with anR:W or an R:F substitution, where the arginine residue is capable ofbeing ADP-ribosylated, is an alpha defensin polypeptide. For example, ifthe alpha defensin polypeptide is HNP-1, the arginine residue atposition 14 of the HNP-1 sequence set forth as SEQ ID NO:1 (or aconservative variant thereof) is capable of being ADP-ribosylated andcan be substituted with a tryptophan residue. In another example, anarginine residue at position 14 is substituted with a phenylalanineresidue.

Cytotoxic activity is measured by the ability of an alpha defensinpolypeptide to lyse a cell. In several embodiments, the lysed cell is anormal cell, a malignant cell, or a cell that is resistant to hostdefense mechanisms. Cell lysis can be measured by any means known todetect the number of viable cells remaining in a sample, following anincubation period. The number of viable cells in the sample is comparedto a control sample. For example, the control can be the number of cellsremaining following incubation with a wildtype alpha defensin.

A method is also provided herein for modulating an activity, such as theNADase activity or a tranferase activity, of an ART. In one embodiment,an R:W substitution and/or an R:F substitution, where the arginineresidue is capable of being ADP-ribosylated, increases the NADaseactivity of the ART. In another embodiment, an R:W substitution and/oran R:F substitution, where the arginine residue is capable of beingADP-ribosylated, increases the transferase activity of the ART. Inseveral specific examples, the ART is ART-1, ART-2a, ART-2b, ART-3,ART-4, and ART-5.

In several specific, non-limiting examples, the increase in NADase orART activity is increased at least about 20%, at least about 50%, atleast about 80%, at least about 100%, or at least about 200%, comparedto a control polypeptide. NADase activity can be measured by any meansknown to one of skill in the art, such as measuring an increase in theamount of hydrolyzed NAD⁺. ART activity can be measured by any meansknown to one of skill in the art, including detecting an increase in theamount ADP-ribosylated arginine residues.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Although ADP-ribosylation of specific residues is known to alter theproperties of modified proteins, the ADP-ribose bond is readily cleavedby pyrophosphatases. Thus, there exists a need to identify additional,stable, protein modifications that have an effect on protein activity.

Mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose fromNAD to one of several specific amino acids in an acceptor protein. Inplace of an amino acid, some of these enzymes utilize water as anacceptor, generating ADP-ribose and nicotinamide from NAD (NADaseactivity). The properties of these enzymes have been most studied in thebacterial toxins (e.g., cholera toxin (CT), an arginine-specificADP-ribosyltransferase (ART)) that uses ADP-ribosylation to modifyproteins that alter activity of critical metabolic or regulatorypathways in mammalian cells (ADP-ribosylating toxins and G proteins:Insights in Signal Transduction (Moss, J., and Vaughan, M., eds.),American Society for Microbiology, Washington, D.C., 1990).

One family of mammalian ARTs have precursor forms with signal sequencesresponsible for export into the ER lumen at the amino termini and insome cases, signal sequences necessary for addition of aglycosylphosphatidylinositol (GPI) anchor, at the carboxy termini(Okazaki et al., J Biol Chem 273(37):23617-20, 1998). Because of theirextracellular localization, these ARTs can potentially regulatecell-cell and cell-matrix interactions through modification of ecto- orextracellular proteins. For example, ART1 modifies integrin α7 in C2C12cells (Zolkiewska et al., J Biol Chem 268(34):25273-6, 1993), andco-receptors of the TCR (e.g., LFA-1, CD8, CD27, CD45) in mouse Tlymphocytes (Nemoto et al., J Immunol 157(8):3341-9, 1996). Thesemodifications, which have been identified in cell culture, requireextracellular NAD at millimolar concentrations. Extracellular proteins,such as defensins that participate in the innate immune response, areADP-ribosylated by ecto-transferases with resulting alteration in theirbiological properties. An ADP-ribosylated HNP-1 was recovered from humanbronchoalveolar lavage fluid consistent with its in vivo modification.An ecto-ART that catalyzes this modification has been identified onhuman airway epithelial cells, suggesting that the airway might utilizean ADP-ribosylation pathway to regulate the immune response (Balducci etal., Am J Respir Cell Mol Biol 21(3):337-46, 1999).

The amino acid sequences of the ARTs differ significantly from those ofthe toxins and each other. ART1 from rabbit skeletal muscle is 30-40%identical in sequence to rat ART2 NADase (RT6) (Balducci et al., Am JRespir Cell Mol Biol 21(3):337-46, 1999). Analysis of thecrystallographic structure of toxin ADP-ribosyltransferases identifiedthree regions involved in formation of the catalytic site, NAD binding,and activation of the ribosyl-nicotinamide bond, which is required forADP-ribose transfer (Domenighini et al., Mol Microbiol 21(4):667-74,1996 and Bredehorst et al., Adv Exp Med Biol 419:185-9, 1997). Theseregions appear to be present in the mammalian transferases as well (Mosset al., Mol Cell Biochem 193(1-2):109-13, 1999). Region I is defined byan arginine (R) or histidine (H), Region II, by a sequence rich inhydrophobic amino acids, or by serine (S) X S, (where X representsthreonine (T), serine (S) or alanine (A)), and Region III by glutamate(E). In ART1 and the bacterial toxins, site-specific mutagenesis ofRegion III verified the importance of Region III glutamate in catalysisand defined a role for a second glutamate in Region III (Takada et al.,J Biol Chem 269(13):9420-3, 1994). Replacement of the second glutamateof human ART1 in the consensus E-X-E sequence abolished activity. Inmouse ART2a (mRt6.1) replacement of the first glutamate with glutamine(Q) abolished the arginine-specific transferase activity giving rise toan NADase (Karsten et al., Adv Exp Med Biol 419:175-80, 1997),suggesting that the carboxyl group of the first glutamate was necessaryfor proper positioning of the guanidino group of arginine. The conversewas true as well: the rat ART2 NADase was converted to anarginine-specific transferase by replacing the first glutamine withglutamate (Hara et al., J Biol Chem 271(47):29552-5, 1996).

Rat ART2b (RT6.2) and ART2a (RT6.1) are encoded by two alleles of asingle copy gene (Thiele et al., Adv Exp Med Biol 419:109-20, 1997); thehuman counterpart has stop signals in the coding region and no proteinis expressed (Haag et al., J Mol Biol 243(3):537-46, 1994). To date,only post-thymic peripheral and intestinal intraepithelial T lymphocytesare known to express ART2 proteins (Mojcik et al., Dev Immunol1(3):191-201, 1991). Both ART2 proteins appear to be released from cellsin vivo and have been found in soluble form in the high-densitylipoprotein fraction of serum (Lesma et al., J Immunol 161(3):1212-9,1998 and Waite et al., Cell Immunol 152(1):82-95, 1993). Although theirbiological functions are unknown, the absence, depletion or reduction ofART2-expressing T lymphocytes is associated with autoimmune diabetes(Bortell et al., Autoimmunity 33(3):199-211, 2001 and Greiner et al., JImmunol 136(1):148-51, 1986). Both proteins are linked to the cellsurface by GPI-anchors, and ART2a, but not ART2b, is glycosylated(Thiele et al., Immunology 59(2):195-201, 1986 and Koch et al.,Immunology 65(2):259-65, 1988). In their mature processed forms, ART2band ART2a differ by ten amino acids. Because of the glutamine in RegionIII (QEE), ART2 catalyzes the hydrolysis of NAD to ADP-ribose andnicotinamide but, in contrast to ART1, does not transfer ADP-ribose toarginine or other small guanidino compounds. The proteins differedsignificantly in their abilities to catalyze auto-modification, withART2b, but not ART2a, capable of auto-ADP-ribosylation at multiplesites. Auto-ADP-ribosylation has been reported to regulate NADase andtransferase activities, most notably those of an erythrocyte NADase, theactivity of which is decreased by auto-ADP-ribosylation (Yamada et al.,Arch Biochem Biophys 308(1):31-6, 1994; Han et al., Biochem J 318(Pt3):903-8, 1996; and Weng et al., J Biol Chem 274(45):31797-803, 1999).

As described in the Examples set forth below, to investigate thestructural requirements for auto-ADP-ribosylation and its effects onactivity, the amino acid sequences of ART2b and ART2a were compared,paying particular attention to the critical catalytic Region III.Replacement of arginine 204 with lysine (R204K) abolished both theprimary and secondary modifications. Replacement with tryptophan,however, (R204W) resulted in auto-ADP-ribosylation at non-argininesites, suggesting that the hydrophobic tryptophan could substitute foran ADP-ribosyl-arginine, consistent with a regulatory role for aminoacid 204 in modification of sites elsewhere in the protein.

Example 1 Materials and Methods

Construction of Wild Type ART2a (RT6.1) and ART2b (RT6.2) ExpressionPlasmids

Rat ART2a open reading frame was amplified by PCR using anART2a-pCRII.1plasmid as a template and cloned in the pMAMneo mammalianexpression vector (Clontech, Palo Alto, Calif.) as an NheI/XhoI fragmentcarrying a Kozak consensus region (GCCACG) upstream of the first codon.Construction of the rat ART2b-pMAMneo mammalian expression vector waspreviously described (Takada et al., J. Biol. Chem. 269:9420-9423,1994). To improve the level of expression of recombinant ART2b inmammalian cells, a Kozak consensus sequence was placed upstream of thefirst ATG, using the QuickChange site-directed mutagenesis kit(Stratagene, La Jolla, Calif.), according to the manufacturer'sinstructions with a pair of complementary mutant primers correspondingto the following sequence:

CGGACTCACCATAGGGACCAAGCTAGCCGCCATGCC ATCAAATATTTGCAAGTTCTTCC (SEQ IDNO:13). Plasmid construct sequences were verified by DNA sequencing ofthe entire open reading frame.

The amino acid sequences of rat ART2b and ART2a differ considerably fromthose of bacterial toxins and other mammalian ADP-ribosyltransferases,although each has large regions of similarity to rabbit ART 1 and thebacterial toxins, particularly, in three regions believed to be involvedin formation of the NAD-binding site. The sequences of ART2b and ART2adiffer by fourteen amino acids, ten of which are located N-terminal tothe region excised during addition of the GPI-anchor (FIG. 1). In ART2b,but not other ARTs, an arginine is present at position 204 (R204) at theamino end of Region III, which contains the consensus glutamate(position 209 in ART2) required for catalytic activity; a putativeconsensus glycosylation site is present at positions 58-60 in ART2a butnot in ART2b.

Site-directed Mutagenesis of Wild Type RT6.1 and RT6.2

Both ART2b and ART2a have NADase activity, but only ART2b issignificantly auto-ADP-ribosylated. To investigate the structural basisfor these differences in catalytic function, site-specific mutagenesiswas employed with synthesis of recombinant ART2b and ART2a proteins inrat adenocarcinoma (NMU) cells under a dexamethasone-sensitive promoter.Point mutations were introduced in ART2a and ART2b cDNAs using theQuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.)according to the manufacturer's instructions. Sequences of sense strandsfrom which pairs of complementary mutation primers were synthesized toproduce the indicated changes in amino acids are indicated in Tables 4and 5, below. Altered bases are underlined.

TABLE 4 Mutation primers for ART2a SEQ Amino acid ID substitutionSequence NO: N58A CCCTGCTTTTAAAGGAAGACTTTGCTAAGAGTGA 14 GAAATTAAAAGTTGCGK59M, S60N, CCCTGCTTTTAAAGGAAGACTTTAATATGAATGC 15 E61A GAAATTAAAAGTTGCGM81R CGATGGAACAACATAAAACCTAGTAGGAGTTATC 16 CCAAAGGTTTCATTGATTTCC Y204RGGGGGTTTATATCAAAGAATTCTCTTTCCGTCCT 17 GACCAAGAGGAGGTG

TABLE 5 Mutation primers for ART2b SEQ Amino acid ID substitutionSequence NO: R81K CGATGGAACAACATAAAACTAGTAAGAGTTATCC 18CAAAGGTTTCAATGATTTC R204K GGGGGTTTATATCAAAGAATTCTCTTTCAAGCCT 19GACCAAGAGGAGGTG R204E GGGGGTTTATATCAAAGAATTCTCTTTCGAGCCT 20GACCAAGAGGAGGTG R204Y GGGGGTTTATATCAAAGAATTCTCTTTCTACCCT 21GACCAAGAGGAGGTG R204W GGGGGTTTATATCAAAGAATTCTCTTTCTGGCCT 22GACCAAGAGGAGGTGAll clones were screened by restriction digestion and confirmed by DNAsequencing (both strands) of the entire open reading frames.Cell Culture and Protein Expression

Rat mammary adenocarcinoma (NMU) cells were grown in Eagle's minimalessential medium with 10% fetal calf serum (GIBCO BRL, Carlsbad, Calif.)at 37° C. in 5% CO₂. Cells were transfected with the pMAMneo vector(Clontech, Palo Alto, Calif.) containing the ART2a or ART2b construct asindicated using the Lipofectomine Plus Reagent (Invitrogen, Carlsbad,Calif.) according to manufacturer's instructions. Transfected cells wereselected with Geneticin (G-418; Life Technologies, Inc, Grand Island,N.Y.), 0.5 mg/ml.

Protein expression was induced with 1 μM dexamethasone (Sigma, St.Louis, Mo.) for 24 hours. Trypsinized confluent cells were sedimented bycentrifugation (1000×g), washed with DPBS, and incubated (1 hour, 37°C.) with 0.05 units of phosphatidylinositol-specific phospholipaseC(PI-PLC) (ICN Pharmaceuticals, Costa Mesa, Calif.) in 500 μl of DPBS tocleave the GPI anchor and release the protein from the cell surface.Cells were sedimented by centrifugation (1000×g), and the supernatantcontaining transferase protein linked to the C-terminal oligosaccharidewas collected.

NAD Glycohydrolase and ADP-ribosyltransferase Assays

NADase activity was measured in DPBS with 0.1 mM [carbonyl-¹⁴C]NAD(8×10⁴ cpm) for 1 hour at 30° C., (total volume=150,u). Samples (50,u)were applied to AG1-X2 (BIO-RAD, Hercules, Calif.) columns (0.4×4 cm),equilibrated and eluted with water for liquid scintillation counting asdescribed (Moss et al., Proc Natl Acad Sci U S A 73(12):4424-7, 1976).Transferase activity was assayed similarly with or without 20 mMagmatine as ADP-ribose acceptor and with [adenine-¹⁴C]NAD substitutedfor [carbonyl-¹⁴C]NAD.

Example 2 Sequence Comparison of ART2b and ART2a

The amino acid sequences of rat ART2b and ART2a differ considerably fromthose of bacterial toxins and other mammalian ADP-ribosyltransferases,although each has large regions of similarity to rabbit ART 1 and thebacterial toxins, particularly, in three regions believed to be involvedin formation of the NAD-binding site. The sequences of ART2b and ART2adiffer by fourteen amino acids, ten of which are located N-terminal tothe region excised during addition of the GPI-anchor (FIG. 1). In ART2bbut not other ARTs, an arginine is present at position 204 (R204) at theamino end of Region III, which contains the consensus glutamate(position 209 in ART2) required for catalytic activity; a putativeconsensus glycosylation site is present at positions 58-60 in ART2a butnot in ART2b.

Example 3 Site Specific Mutagenesis Amino Acid Substitutions

Both ART2b and ART2a have NADase activity, but only ART2b issignificantly auto-ADP-ribosylated. To investigate the structural basisfor these differences in catalytic function, site-specific mutagenesiswas employed with synthesis of recombinant ART2b and ART2a proteins inrat adenocarcinoma cells (NMU) under a dexamethasone-sensitive promoter.After induction and expression of the GPI-anchored proteins, cells wereincubated with PI-PLC to cleave the GPI anchor and then [³²P]-NAD wasadded to the medium to promote auto-ADP-ribosylation of the releasedprotein (FIG. 2). All recombinant proteins had NADase activity and allwere reactive with antipeptide antisera NAD2, exhibiting the expectedsize of 29 kDa on immunoblots. ART2a, the glycosylated isoform, had anadditional band at approximately 33 kDa, consistent with its singleconsensus sequence for N-glycosylation.

Multiple species of auto-ADP-ribosylated wild-type ART2b were observedby SDS-PAGE. Substitution of lysine for arginine 81 had no effect onauto-ADP-ribosylation whereas it was abolished by replacement ofarginine-204 by lysine (R204K) (FIG. 2), consistent with arginine-204being the primary modification site. As expected (and shown in FIG. 5),the ADP-ribosylarginine bond was sensitive to hydroxylamine. Replacementof R204 with Y, E and K in ART2b(R81K) (FIG. 3) abolishedauto-ADP-ribosylation, although all proteins retained NADase activity.Wild-type ART2a with tyrosine in position 204 was not significantlyauto-modified. However, in ART2a(Y204R) or ART2a(M81R,Y204R),auto-ADP-ribosylation of both glycosylated 33 kDa and nonglycosylated 29kDa forms was observed (FIG. 3). The substitution of arginine atposition 81 alone, ART2a(M81R), had no effect.

NADase activities of all ART2a mutants were lower than that of wild-typeART2a, independent of their ability to be auto-ADP-ribosylated. Theseresults were consistent with a role for position 204 in regulatingNADase activity. In ART2a(Y204R), modification of the putative consensusglycosylation site, replacing N58 with A, or changing 59KSE61 to59MNA61, prevented glycosylation, resulting in a single immunoreactiveband after SDS-PAGE. Both non-glycosylated species wereauto-ADP-ribosylated. Thus, a single amino acid, R204, is responsiblefor auto-ADP-ribosylation of ART2b or ART2a(Y204R).

Example 4 Auto-ADP-Ribosylation

To assess the ability of ART2 proteins, ART2a and ART2b, to beauto-ADP-ribosylated at multiple sites, proteins were incubated firstwith 10 μM [³²P]NAD, followed by addition of 5 mM unlabeled NAD andfurther incubation (FIG. 4). Wildtype ART2b, ART2b(R81K) and mutantsART2a(Y204R), ART2a(Y204R,M81R) and ART2a(59MNA61,Y204R), exhibitedauto-ADP-ribosylation following incubation with 5 mM NAD as evidenced bya decrease in mobility on SDS-PAGE, consistent with modification ofmultiple sites. In contrast, ART2b(R204K) was not modified by incubationwith NAD, suggesting that primary and secondary auto-ADP-ribosylationsites were lost (FIG. 5). It is unlikely that auto-modification was dueto non-enzymatic addition of [³²P]ADP-ribose since reactions werecarried out in the presence of 1 mM ADP-ribose, or due to NAD binding,since the radiolabel was not displaced by incubation with 5 mM NAD.

Thus, arginine at position 204 regulates the auto-ADP-ribosylation ofmultiple sites in ART2b and ART2a(Y204R). Since lysine, a conservativesubstitution, could not replace arginine, ADP-ribosylation of arginineappears to be an initiating event required for modifications elsewherein the protein. When multiple auto-ADP-ribosylation sites were seen onSDS-PAGE, a decrease in the rate of NAD hydrolysis by wildtype ART2b wasobserved. The activity of wildtype ART2b decreased during the course ofthe reaction, as did ART2b(R204W), while that of ART2b(R204K), which isnot auto-ADP-ribosylated, did not change.

These data suggest that the residues which are auto-modified have accessto the catalytic site, and hence, can be modified, can regulateauto-ADP-ribosylation activity as in the case of ART2a(Y204R), and canmodulate NADase activity. Without being bound by theory, one possiblereason for this in ART2a is because of the proximity of the residues toRegion III glutamate. All ART2a mutants capable of auto-ADP-ribosylation(i.e., arginine at position 204) had reduced NADase activity compared towild-type (FIG. 3).

Example 5 Ribosylation of ART2b(R204K) and Characterization ofADP-Ribose Bonds

To investigate whether ART2b(R204K) could be modified, wildtype ART2bwas incubated with its mutant ART2b(R204K) and millimolar NAD (FIG. 5).Following incubation with NAD with or without ART2b(R204K), ART2bexhibited decreasing mobility on SDS-PAGE, indicating that it wasauto-ADP-ribosylated. The migration of ART2b(R204K), however, wasunchanged, consistent with the conclusion that ART2b(R204K) is notmodified by wildtype ART2b in an intermolecular reaction.

An ADP-ribose-amino acid linkage can be characterized by its sensitivityto acid, hydroxylamine, and mercuric chloride (FIG. 6). To characterizethe multiple ADP-ribose bonds that result from incubation of wildtypeART2b, or ART2a(M81R,Y204R) with millimolar NAD, their chemicalsensitivity was tested. The PI-PLC released proteins were incubated with10 μM [³²P]NAD, [RT6.1(N58A, Y204R) and RT6.2(R204W)], or also followedby 5 mM NAD. For auto-ADP-ribosylated ART2b, ART2a(M81R, Y204R) andRT6.1(N58A,Y204R), hydroxylamine released the [³²P]ADP-riboseradiolabel, consistent with the chemical stability of anADP-ribose-arginine linkage.

Surprisingly, as disclosed herein, a mutant ART2b with tryptophanreplacing arginine-204, ART2b(R204W), had weak auto-ADP-ribosylationactivity at multiple sites with NADase activity greater than that ofwild-type ART2b (FIGS. 3,4). [³²P]ADP-ribose was not released fromauto-ADP-ribosylated ART2b(R204W) by hydroxylamine, mercury chloride oracid suggesting that arginine, cysteine, and lysine respectively werenot modified by the auto-ADP-ribosylation reaction (FIG. 6). Thus, thetryptophan mutant is an auto-ADP-ribosyltransferase although it differsfrom wild-type ART2b in the ADP-ribose acceptor site(s). Without beingbound by theory, the tryptophan may function in the regulatory role ofan ADP-ribosylated arginine-204. The bulky side chain or hydrophobicityof tryptophan coupled with its position in Region III in proximity tothe catalytic glutamate (amino acid 209) could promoteauto-ADP-ribosyltransferase, as well as NADase activity. The mutant,like the wildtype, however, is unable to transfer ADP-ribose toagmatine.

The ability of tryptophan to replace some of the function ofADP-ribosylarginine is likely to be of use in protein design. AlthoughADP-ribosylation has effects on protein function, the modificationitself is unstable in biological systems. It can be cleaved bypyrophosphatases, with release of AMP, and the resulting phospho-ribosylprotein, further degraded by phosphatases, yielding ribosyl-protein.Obviously, synthesis of ADP-ribosylated proteins requires an additionalstep(s) following production of a recombinant molecule. In contrast, aprotein containing tryptophan can be produced by standard techniques andshould have no unusual instability in biological systems. As disclosedherein, replacement of an arginine that is capable of being ribosylatedwith a tryptophan or a phenylalanine finds use in many systems,including, but not limited to, the defensins, as described below.

Example 6 Cytotoxicity Assay

The antibacterial activity of different concentrations (16, 32, 64, 128,256 nM) of HNP-1, ADP-ribosyl-HNP-1, or HNP-1 with either a R:Wsubstitution or a R:F substitution, on Escherichia coli ATCC43827(American Type Culture Collection, Rockville, Md.) is evaluated by theradial diffusion assay (Takemura et al., “Evaluation of susceptibilityof gram positive and negative bacteria to human defensins by usingradial diffusion assay,” Antimicrob. Agents Chemother 40:2280-2284,1996). The results indicate that HNP-1 with an R:W substitution or anR:F substitution behaves in a manner similar to ADP-ribosylated HNP-1,which is less cytotoxic than unmodified HNP-1 for E. coli ATCC43827 inthe radial diffusion assay (see PCT Application No. PCT/US03/04649,which is incorporated herein by reference).

Example 7 Chromium Release Assay

Chromium-labeled A549 cells (American Type Culture Collection) wereincubated (18 hours, 37° C.) in 100 μl of serum-free RPMI (Gibco FluidsInc., Rockville, Md.) containing HNP-1, ADP-ribosyl-HNP-1, or HNP-1 witheither an R:W substitution or an R:F substitution (1.5 to 24 μM) toquantify defensin cytotoxicity. Cytotoxicity is measured as the amountof chromium released from the cells (Panyutich et al., “Human neutrophildefensins and serpins form complexes and inactivate each other,” Am. J.Respir. Cell. Mol. Biol. 12:351-357, 1995). The results indicate thatHNP-1 with either an R:W substitution or an R:F substitution andADP-ribosylated HNP-1 are less cytotoxic than unmodified HNP-1 for A549cells.

Example 8 Radial Diffusion and Chromium Release Assays

HNP-1 (100 nM) is incubated for 1 hour at 37° C. with ADP-ribosylatedHNP-1, or HNP-1 with either an R:W substitution or an R:F substitution(0-800 nM) before the initiation of the cytotoxicity assay on E. coli.

HNP-1 (12 μM) is incubated with ADP-ribosyl-HNP-1, or HNP-1 with eitheran R:W substitution or an R:F substitution (1.5-12 μM) for 1 hour at 37°C. before the initiation of the chromium release assay.

ADP-ribosylated HNP-1 blocks the cytotoxic activity of HNP-1 in aconcentration-dependent manner. HNP-1 with either an R:W substitution oran R:F substitution has the same effect on HNP-1 as ADP-ribosylatedHNP-1.

Example 9 IL-8 Production by A549 Cells

A549 cells (3×10⁴ cells per well) are incubated in a 96-well plate in200 μl of serum-free RPMI medium (Gibco Fluids Inc) containingADP-ribosyl-HNP-1, HNP-1, or HNP-1 with either an R:W substitution or anR:F substitution (0.25, 0.75, 1.5, 3 mM). Culture medium is sampledafter 12 or 24 hours of incubation and IL-8 content is assayed byindirect ELISA according to the manufacturer's instructions (R & DSystem Inc. Minneapolis, Minn.). At concentrations of 0.75 and 1.5 μM,IL-8 release is significantly higher with ADP-ribosylated HNP-1, or withHNP-1 with either an R:W substitution or an R:F substitution, than withthe unmodified peptide.

Example 10 Chemotaxis Assay

CD3⁺ T-cells are isolated from human peripheral blood prepared byleukapheresis (NIH, Department of Transfusion Medicine, Bethesda, Md.)(Chertov et al., J. Biol. Chem. 271:2935-2940, 1996) and suspended inmigration medium (RPMI 1640, 0.5% bovine serum albumin, 25 mM HEPES).Inserts coated with collagen IV (Becton Dickinson Labware, Bedford,Mass.) are placed into 24-well culture plates to form upper and lowerchambers in each well. Upper chambers are wetted with migration medium,then 500 μl of migration medium with or without ADP-ribosyl-HNP-1,HNP-1, or with HNP-1 with either an R:W substitution or an R:Fsubstitution (0.025 to 25 nM) are added to the lower chamber. Cells areadded to the upper chamber and plates are incubated at 37° C. in 5% CO₂for 4 hours. Lymphocytes that migrate to the lower chamber are harvestedby centrifugation and counted in a hematocytometer. The results indicatethat ADP-ribosyl HNP-1 and HNP-1 with either an R:W substitution or anR:F substitution retain their ability to recruit T cells.

This disclosure provides methods of producing polypeptides with arginineresidues that are replaced with tryptophan or phenylalanine residuesresulting in polypeptide with increased activity and/or stability. Thedisclosure further provides methods of screening for polypeptides thatcan be stabilized, as well as methods for modifying the activity of apolypeptide. It will be apparent that the precise details of the methodsdescribed may be varied or modified without departing from the spirit ofthe described invention. We claim all such modifications and variationsthat fall within the scope and spirit of the claims below.

1. A substituted human defensin polypeptide, comprising : (i) atryptophan residue or a phenylalanine residue substituted for at leastone arginine residue in a corresponding non-substituted humanalpha-defensin or human beta-defensin polypeptide; (ii) six conservedcysteine residues that form three disulfide bonds; (iii) decreasedcytotoxic activity, compared to the corresponding non-substituted humandefensin polypeptide; and (iv) increased polypeptide stability, comparedto the corresponding non-substituted human defensin polypeptide, whereinthe at least one arginine residue in the corresponding non-substitutedhuman defensin polypeptide is ADP-ribosylated, wherein the polypeptideis no more than 94 amino acids in length and retains anti-microbialactivity.
 2. The substituted human defensin polypeptide of claim 1,wherein the polypeptide is no more than 50 amino acids in length.
 3. Thesubstituted human defensin polypeptide of claim 1, wherein a tryptophanresidue is substituted for the at least one arginine residue.
 4. Thesubstituted human defensin polypeptide of claim 1, wherein aphenylalanine residue is substituted for the at least one arginineresidue.
 5. The substituted human defensin polypeptide of claim 2,wherein the polypeptide is between 29 and 34 or between 34 and 37 aminoacids in length.
 6. The substituted human defensin polypeptide of claim1, wherein the corresponding non-substituted human defensin is an alphadefensin.
 7. The substituted human defensin polypeptide of claim 6,wherein the alpha defensin is human neutrophil peptide (HNP)-1, HNP-2,HNP-3, HNP-4, human defensin (HD)-5, HD-6, or defensin-X.
 8. Apharmaceutical composition comprising a therapeutically effective amountof the substituted human defensin polypeptide of claim
 1. 9. Thesubstituted human defensin polypeptide of claim 1, wherein theantimicrobial activity comprises chemotaxis of T cells, neutrophilrecruitment or cytokine release.
 10. The pharmaceutical composition ofclaim 8, wherein the antimicrobial activity comprises chemotaxis of Tcells, neutrophil recruitment or cytokine release.
 11. The substitutedhuman defensin polypeptide of claim 7, wherein the substituted humandefensin polypeptide comprises an amino acid sequence set forth as SEQID NO: 3 and wherein at least one arginine residue at position 4, 13,14, or 23 of SEQ ID NO: 3 is substituted with a tryptophan residue or aphenylalanine residue.
 12. The substituted human defensin polypeptide ofclaim 7, wherein the substituted human defensin polypeptide comprises anamino acid sequence set forth as SEQ ID NO: 6 and wherein at least onearginine residue at position 5, 10, 11, 15, or 32 of SEQ ID NO: 6 issubstituted with a tryptophan residue or a phenylalanine residue. 13.The substituted human defensin polypeptide of claim 7, wherein thesubstituted human defensin polypeptide comprises an amino acid sequenceset forth as SEQ ID NO: 8 and wherein at least one arginine residue atposition 5, 8, 12, 24, 27, or 31 of SEQ ID NO: 8 is substituted with atryptophan residue or a phenylalanine residue.
 14. The substituted humandefensin polypeptide of claim 7, wherein the substituted human defensinpolypeptide comprises an amino acid sequence set forth as SEQ ID NO: 10and wherein at least one arginine residue at position 5, 6, or 26 of SEQID NO: 10 is substituted with a tryptophan residue or a phenylalanineresidue.
 15. The pharmaceutical composition of claim 8, wherein thesubstituted human defensin polypeptide comprises an amino acid sequenceset forth as SEQ ID NO: 3 and wherein at least one arginine residue atposition 4, 13, 14, or 23 of SEQ ID NO: 3 is substituted with atryptophan residue or a phenylalanine residue.
 16. The pharmaceuticalcomposition of claim 8, wherein the substituted human defensinpolypeptide comprises an amino acid sequence set forth as SEQ ID NO: 6and wherein at least one arginine residue at position 5, 10, 11, 15, or32 of SEQ ID NO: 6 is substituted with a tryptophan residue or aphenylalanine residue.
 17. The pharmaceutical composition of claim 8,wherein the substituted human defensin polypeptide comprises an aminoacid sequence set forth as SEQ ID NO: 8 and wherein at least onearginine residue at position 5, 8, 12, 24, 27, or 31 of SEQ ID NO 8 issubstituted with a tryptophan residue or a phenylalanine residue. 18.The pharmaceutical composition of claim 8, wherein the substituted humandefensin polypeptide comprises an amino acid sequence set forth as SEQID NO: 10 and wherein at least one arginine residue at position 5, 6, or26 of SEQ ID NO: 10 is substituted with a tryptophan residue or aphenylalanine residue.
 19. The substituted human defensin polypeptide ofclaim 1, wherein the corresponding non-substituted human defensin is abeta defensin.
 20. The substituted human defensin polypeptide of claim19, wherein the beta defensin is human beta defensinl (hBD1), hBD2,hBD3, or hBD4.
 21. The substituted human defensin polypeptide of claim11, wherein the substituted human defensin polypeptide comprises anamino acid sequence set forth as SEQ ID NO: 2 and wherein at least onearginine residue at position 5, 14, 15, or 24 of SEQ ID NO: 2 issubstituted with a tryptophan residue or a phenylalanine residue. 22.The substituted human defensin polypeptide of claim 11, wherein thesubstituted human defensin polypeptide comprises an amino acid sequenceset forth as SEQ ID NO: 4 and wherein at least one arginine residue atposition 5, 14, 15, or 24 of SEQ ID NO: 4 is substituted with atryptophan residue or a phenylalanine residue.
 23. The substituted humandefensin polypeptide of claim 11, wherein the substituted human defensinpolypeptide comprises an amino acid sequence set forth as SEQ ID NO: 3and wherein the arginine residue at position 13 of SEQ ID NO: 3 issubstituted with a phenylalanine residue.